抗大鲵虹彩病毒MCPCOE蛋白单克隆抗体的制备及初步应用  

Preparation and preliminary application of a monoclonal antibody against MCP COE protein of Andrias davidianus iridovirus

作　　者：周小愿[1] 张星朗[1] 贾秋红[1] 韩亚慧 高宏伟[1] 

机构地区：[1]中国水产科学研究院黄河水产研究所,陕西西安710086

出　　处：《水产学报》2016年第12期1923-1930,共8页Journal of Fisheries of China

基　　金：陕西省科学技术发展计划项目(2012K01-18;2014k01-20-02)~~

摘　　要：制备抗大鲵虹彩病毒(CGSIV)MCP COE蛋白的单克隆抗体,并对其基本特性进行鉴定及开展初步应用。用纯化的重组CGSIV MCP COE蛋白免疫BALB/c小鼠,取免疫小鼠脾细胞与SP2/0骨髓瘤细胞融合,间接ELISA法检测阳性孔,经有限稀释法亚克隆,筛选单克隆杂交瘤细胞株。采用间接ELISA法、免疫印迹法和单克隆抗体亚型检测试剂盒等鉴定单抗的稳定性、特异性和亚型;采用腹水诱生法制备单抗腹水,饱和硫酸铵法纯化腹水单抗;间接ELISA法分别测定单抗杂交瘤细胞上清液和腹水单抗的效价;利用间接免疫荧光法观察CGSIV的增殖。结果显示,细胞融合克隆率为98.68%,阳性率为20.52%,得到1株能够稳定分泌特异性抗体的阳性杂交瘤细胞,命名为1M6。单克隆抗体的亚型属于Ig G2b,κ链;免疫印迹法和间接ELISA法测定结果显示,单抗具有很好的特异性;杂交瘤细胞株培养上清液的效价为1∶1600,腹水单抗效价为1∶2×106,亲和常数为2×105。间接免疫荧光显示CGSIV感染EPC细胞24 h后在宿主细胞质中可以观察到成熟的病毒粒子形成的包涵体,而且在宿主细胞核内也能发现病毒粒子。抗CGSIV MCP COE蛋白单克隆抗体的成功制备为CGSIV免疫学检测方法的建立和其他相关研究的开展奠定了基础。This work was to prepare and characterize a monoclonal antibody against MCP COE protein of Chinese giant salamander（Andrias davidianus） iridovirus（CGSIV）,so as to provide a valuable tool for CGSIV diagnosis and further researches on CGSIV pathogenesis. After being purified by nitrilotriacetic acid（Ni-NTA） agarose resin,the recombinant MCP COE of CGSIV was used to immunize the BALB/c mice. Three days after the last immunization,spleen cells were removed and fused with myeloma cells. One hybridoma cell strain against the recombinant protein of MCP COE was obtained by screening with the indirect ELISA and limiting dilution assays,which was named 1M6. The Mc Ab of 1M6 was identified to be Ig G2 b isotype with κ light chain and showed specific reactivity with CGSIV and the recombinant protein of MCP COE. The Mc Ab was largely produced in mouse ascites and then depurated with saturated ammonium sulfate. Indirect ELISA assay showed that the valences of the Mc Abs in the cells culture supertanant and in mouse ascites were 1∶1600 and 1∶2×106respectively. Affinity constant of the Mc Ab of 1M6 was 2×105. By immunofluorescence assay,the presence of CGSIV was observed in not only the cytoplasm but also the nucleus of CGSIV infected EPC cells. In conclusion,the prepared Mc Ab has high titer and good specificity,which will lay the foundation for further studies on the control of CGSIV.

关 键 词：大鲵 虹彩病毒 主要衣壳蛋白 单克隆抗体 

分 类 号：Q511[生物学—生物化学] S947.3[农业科学—水产养殖]